Recombinant attenuated clostridium organisms and vaccine

ABSTRACT

The present invention discloses attenuated  Clostridium perfringens  organisms that express a substantially nontoxic alpha-toxin. The expressed alpha-toxin is a deletion mutein that relative to the alpha-toxin of the mature alpha-toxin of  Clostridium perfringens  strain 13, is missing at least nine consecutive amino acid residues including His 68 . The present invention also discloses attenuated organisms that encode the muteins, as well as the use of such attenuated organisms as vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application that claims priorityunder 35 U.S.C. § 119(e) of provisional applications U.S. Ser. No.60/792,553 filed Apr. 17, 2006, the contents of which are herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention is related to attenuated Clostridium organisms, methodsof making and using the same, and mutein alpha-toxins and nucleic acidsencoding the same.

BACKGROUND OF THE INVENTION

Anaerobic bacterial pathogens are a serious economic burden on theagricultural industry. Bacteria of the Clostridium family represent aparticular burden, because these bacteria cause serious diseases inpoultry and other economically valuable domestic animals. Previousefforts to control these organisms have relied upon sanitary measuresand the administration of antibiotics in the animal feed.

In particular, Clostridium perfringens (“C. perfringens”) is ananaerobic bacterium that is found in the soil, decaying organic matter,and as part of the gut flora of humans and animals. Different strains ofC. perfringens are designated as biotypes A through E, depending on thespectrum of toxins produced [Justin et al., Biochemistry 41, 6253-6262(2002); McDonel (1986) PHARMACOLOGY OF BACTERIAL TOXINS; F Dorner and JDrews (Eds.) Pergamon Press, Oxford]. Biotype A strains are ofparticular importance as the etiological agents of various types ofgangrene and enteric diseases. A particularly serious enteric diseasecaused by C. perfringens is enteritis necroticans (also art-known as,“necrotic enteritis”), a gangrene of the intestines resulting innecrosis, sepsis, and hemolysis, in both humans and domesticated animals[see, Pearson et al., J. Am. Vet. Med. 188(11):1309-10 (1986);Al-Sheikhy and Truscott, Avian Dis. 21(2):256-63 (1977)]. For avians,e.g., chickens (Gallus gallus), enteritis necroticans is a significantproblem. C. perfringens of either type A or type C can cause majorlosses, especially in production broiler chickens [Ficken and Wages,Necrotic Enteritis, In Diseases of Poultry, 10th Ed. pps 261-264(1997)]. In addition to losses associated with necrotic enteritisoutbreaks, productivity is reported to be impaired in flocks with C.perfringens-associated disease [Lovl and Kaldhusdal, Avian Pathology30:73-81 (2001)]. As noted above, antibacterial agents inserted in theanimal feed are the most common method of control. However,antibacterial agents, e.g., antibiotics, are costly and subject toincreasing concerns related to the promotion of bacterial resistance.

More recently, attempts have been made to provide vaccines againstharmful Clostridium species. For example, Lovland et al. [AvianPathology 33(1):83-92 (2004)] demonstrated candidate vaccines based onC. perfringens type A and type C toxoids with an aluminum hydroxideadjuvant. Vaccination of parent hens was reported to provide specificantibodies to protect progeny against enteric lesions induced bysubclinical challenge with C. perfringens. Other toxoid-based vaccinesprepared from detoxified C. perfringens toxins are known [see e.g., U.S.Pat. No. 4,292,307, which describes toxoids of C. perfringens types A, Band D, Cl. oedematiens, and Cl. septicum].

Recombinant toxoid preparations also have been proposed. For example,Titball et al., [U.S. Pat. Nos. 5,851,827, 6,403,094, and 5,817,317]report nucleic acids that encode antigenic C. perfringens peptides, aswell as the peptides themselves, and vaccines prepared from thepeptides. Peptides are described for example, which have amino acidresidues 261 to 300 of the natural C. perfringens alpha-toxin, but lackthe phosphoplipase C and sphinogmyelin hydrolyzing domains of thenatural toxin. It was further reported that these peptides induce immuneprotection against the natural toxin. In addition, U.S. Pat. No.6,610,300 describes a vaccine based on an antigenic fragment of a muteinC. perfringens beta-toxin.

However, no matter whether a toxoid vaccine is derived from the nativeorganism or is obtained recombinantly, it is considered to beeconomically burdensome to produce and administer toxoid proteins toanimals in need of immunization, except under special circumstances(e.g., treating humans who might be allergic or sensitive to othercomponents of a whole organism vaccine). Further, protein/toxoidbased-vaccines typically require repeated booster vaccinations in orderto maintain full effectiveness.

Another proposed solution has been to engineer an antigenically activevirus that will produce a mutein alpha-toxin, in place of the wild-typetoxin. For example, Bennett et al. [Viral Immunol. 12(2):97-105 (1999)]have demonstrated a recombinant vaccinia virus vector that expresses anontoxic C-domain of C. perfringens alpha-toxin. Unfortunately, whileseveral recombinant vaccinia vaccines have been proposed during the past20 years, there are still longstanding concerns about the safety ofreleasing live, infectious vaccinia viruses into an environment wherethey might be transmitted to those people who are not resistant to thisvirus.

The alpha-toxin (plc gene) of C. perfringens is known to possess severalbiological activities including hemolytic activity, phospholipase C,sphingomyelinase, phosphodiesterase, and lethal activities. There are anumber of reports in the art concerning mutations to this alpha-toxinthat reduce toxicity. Schoepe, et al. [Infect. and Immun. 69(11):7194-7196 (2001)] describe a naturally-occurring C. perfringens strainthat produces a non-toxic alpha-toxin. However, it would be difficult tomodify this strain to elicit immune protection against other variant,but toxic wild-type C. perfringens species.

Williamson and Titball [Vaccine 11(12):1253-1258 (1993)] showed that theregion of the toxin from amino acid residues 247 to 370 alone wassufficient to immunize mice against gas gangrene experimentally inducedby C. perfringens. Alape-Girón et al. [Eur. J. Biochem. 267:5191-5197(2000)] have reported that substitutions in Asp269, Asp336, Tyr275,Tyr307, and Tyr331 reduced alpha-toxin toxicity. Nagahama, et al.[Infect. and Immun. 65:3489-3492 (1997)] reported that replacement ofAsp-56, Asp-130, or Glu-152 resulted in reduced alpha-toxin toxicity.Nagahama et al. [J. Bacteriology 177:1179-1185 (1995)] reported thatsubstitution of the histidine at position 68 with a neutral amino acid,such as glycine, in the C. perfringens alpha-toxin resulted in acomplete loss of hemolytic, phospholipase C, sphingomyelinase, andlethal activity of the mutein alpha-toxin. This single amino acid changewas believed to inactivate one of the three zinc-binding domains of theprotein. The zinc-binding domain inactivated by substitution of His68was later denoted as Zn2 [Justin et al., Biochemistry 41:6253-6262(2002)].

Despite the foregoing, there remains a need in the art for a safe,economical and effective method of protecting intensively cultivateddomestic animals, including avians, such as chickens, from infection byClostridium species, including C. perfringens.

The citation of any reference herein should not be construed as anadmission that such reference is available as “prior art” to the instantapplication.

SUMMARY OF THE INVENTION

In order to address the above-described shortcomings in the art, thepresent invention provides nucleic acid molecules that encode asubstantially nontoxic mutein of Clostridium perfringens alpha-toxin. Inone such embodiment, the nucleic acid molecule encodes a muteinalpha-toxin that comprises the amino acid sequence of SEQ ID NO: 3,minus at least 18 consecutive amino acid residues, wherein one of thedeleted amino acid residues is His₆₈. In another embodiment, the nucleicacid molecule encodes a mutein alpha-toxin that comprises the amino acidsequence of SEQ ID NO: 3, minus at least 12 consecutive amino acidresidues, wherein one of the deleted amino acid residues is His₆₈. Instill another embodiment, the nucleic acid molecule encodes a muteinthat comprises the amino acid sequence of SEQ ID NO: 3 minus at least 9consecutive amino acid residues, wherein one of the deleted amino acidresidues is His₆₈. In yet another embodiment, the nucleic acid moleculeencodes a mutein that comprises the amino acid sequence of SEQ ID NO: 3minus at least 6 consecutive amino acid residues, wherein one of thedeleted amino acid residues is His₆₈. In still another embodiment, thenucleic acid molecule encodes a mutein that comprises the amino acidsequence of SEQ ID NO: 3 minus at least 3 consecutive amino acidresidues, wherein one of the deleted amino acid residues is His₆₈.

In one embodiment, a nucleic acid molecule of the present inventionencodes a mutein in which no more than 48 consecutive amino acidresidues are deleted from the amino acid sequence of SEQ ID NO: 3. Inanother embodiment, a nucleic acid molecule encodes a mutein in which nomore than 36 consecutive amino acid residues are deleted from the aminoacid sequence of SEQ ID NO: 3. In yet another embodiment a nucleic acidmolecule encodes a mutein in which no more than 24 consecutive aminoacid residues are deleted from the amino acid sequence of SEQ ID NO: 3.In still another embodiment a nucleic acid molecule of the presentinvention encodes a mutein in which no more than 18 consecutive aminoacid residues are deleted from the amino acid sequence of SEQ ID NO: 3.

In a particular embodiment, the present invention provides a nucleicacid molecule encoding a mutein in which nine consecutive amino acidresidues are deleted from the amino acid sequence of SEQ ID NO: 3, oneof which is His₆₈. In a particular embodiment of this type, the nucleicacid molecule encodes a mutein in which the deleted nine consecutiveamino acid residues range from Tyr₆₂ through Trp₇₀ of SEQ ID NO: 3. In amore particular embodiment, the nucleic acid molecule encodes a muteinin which these deleted nine consecutive amino acids are replaced by asingle leucine residue.

In another embodiment, the nucleic acid molecule comprises thenucleotide sequence of SEQ ID NO: 2, wherein nucleotides 268-294 aredeleted. In a specific embodiment of this type, nucleotides 268-294 ofthe nucleotide sequence of SEQ ID NO:2 are replaced by three nucleotidesthat encode a single leucine residue.

The invention also provides substantially nontoxic muteins ofClostridium perfringens alpha-toxin. In one such embodiment the muteinalpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus atleast 18 consecutive amino acid residues, wherein one of the deletedamino acid residues is His₆₈. In another embodiment, the muteincomprises the amino acid sequence of SEQ ID NO: 3 minus at least 12consecutive amino acid residues, wherein one of the deleted amino acidresidues is His₆₈. In still another embodiment, the mutein comprises theamino acid sequence of SEQ ID NO: 3 minus at least 9 consecutive aminoacid residues, in which one of the deleted amino acid residues is His₆₈.In yet another embodiment, the mutein comprises the amino acid sequenceof SEQ ID NO: 3 minus at least 6 consecutive amino acid residues,wherein one of the deleted amino acid residues is His₆₈. In stillanother embodiment, the mutein comprises the amino acid sequence of SEQID NO: 3 minus at least 3 consecutive amino acid residues, wherein oneof the deleted amino acid residues is His₆₈.

In one embodiment, a mutein of the present invention comprises no morethan 48 consecutive amino acid residues that are deleted from the aminoacid sequence of SEQ ID NO: 3. In another embodiment, the muteincomprises no more than 36 consecutive amino acid residues deleted fromthe amino acid sequence of SEQ ID NO: 3. In yet another embodiment themutein comprises no more than 24 consecutive amino acid residues deletedfrom the amino acid sequence of SEQ ID NO: 3. In still anotherembodiment the mutein comprises no more than 18 consecutive amino acidresidues deleted from the amino acid sequence of SEQ ID NO: 3.

In a particular embodiment, the present invention provides asubstantially nontoxic mutein in which nine consecutive amino acidresidues are deleted from the amino acid sequence of SEQ ID NO: 3, oneof which is His₆₈. In a more particular embodiment of this type, thedeleted nine consecutive amino acid residues range from Tyr₆₂ throughTrp₇₀ of SEQ ID NO: 3. In still a more particular embodiment, thesedeleted nine consecutive amino acids are replaced by a single leucineresidue in the amino acid sequence of the mutein.

The invention further provides attenuated Clostridium perfringensorganisms that have a nucleic acid molecule that encodes a substantiallynontoxic mutein of Clostridium perfringens alpha-toxin integrated intotheir chromosomes. This integrated nucleic acid molecule is preferablylocated at a position on the chromosome that is homologous to thelocation of the nucleic acid molecule that encodes the wild-typealpha-toxin in the wild-type Clostridium perfringens organism. Thus, anattenuated Clostridium perfringens organism of the present invention canbe substantially nontoxic due to the lack of a functional wild-type plcgene. As exemplified herein, the attenuated Clostridium perfringensorganism can be a type A Clostridium perfringens.

In a particular embodiment of the present invention, the attenuatedClostridium perfringens organism is Clostridium perfringensCPERF/ΔαToxin 365-054 (ATCC Deposit No. PTA7364). In another particularembodiment of the present invention, the attenuated Clostridiumperfringens organism is Clostridium perfringens CPERF/ΔαToxin 365-053(ATCC Deposit No. PTA7365).

A Clostridium perfringens organism that is attenuated by the methods ofthe present invention can be isolated from a host animal that is eithera mammal or an avian. Such mammals can include: bovine, ovine, andporcine. Examples of appropriate avians include chickens, turkeys,ducks, pigeons, geese, doves, swans, partridge, and grouse.

The present invention also provides vaccines. Such vaccines can comprisethe attenuated Clostridium perfringens organisms of the presentinvention. The vaccines of the prevent invention can also includepharmacologically acceptable buffers, excipients, and/or adjuvants.

In addition, the invention provides methods of inducing immunity toClostridium perfringens in an animal. One such embodiment comprisesadministering an immunologically effective dose of a vaccine of thepresent invention to the animal. The vaccines of the present inventioncan be administered by a number of routes including: orally,intramuscularly, intravenously, intradermally, subcutaneously andintranasally. A vaccine of the present invention can be top-dressed onthe feed of the animal and/or sprayed onto the animals to provide fororal administration. The present invention further provides an animalfeed that includes a vaccine of the present invention.

The present invention also provides an attenuated Clostridiumperfringens organism of the present invention that in addition,expresses at least one gene encoding a non-Clostridium perfringenspolypeptide. In one such embodiment one or more of the non-Clostridiumperfringens polypeptides are bacterial polypeptides, such as antigenicproteins from E. coli, salmonella, lawsonia, or campylobacter etc.,and/or combinations thereof. Alternatively, or in combination therewith,a non-Clostridium perfringens polypeptide can be a non-bacterialpolypeptide. Examples of such non-bacterial polypeptides includemammalian or avian proteins, e.g. cytokines, such as chicken IL-18;viruses such as rotavirus or coronavirus; and parasites such as eimeria,isospora, and cryptosporidium.

In another aspect, the present invention provides an antibody thatselectively binds to an epitope missing from the substantially nontoxicmutein of Clostridium perfringens alpha-toxin. Such antibodies candistinguish the substantially nontoxic mutein from a wild-typeClostridium perfringens alpha-toxin.

Test kits are also provided that include the antibodies of the presentinvention for use in identifying whether a subject animal has beenvaccinated, or alternatively, has been naturally infected by aClostridium perfringens organism.

Accordingly, methods of identifying and/or distinguishing an animal thathas been naturally infected by a Clostridium perfringens organism fromone vaccinated with an attenuated Clostridium perfringens organism arealso provided. In one such embodiment the method entails contacting afluid sample from the animal with an antibody that selectively binds toan epitope found in a wild type Clostridium perfringens alpha-toxin thathas been deleted from the substantially nontoxic mutein of Clostridiumperfringens alpha-toxin of the present invention. Therefore, theantibody can distinguish those animals that have been vaccinated with asubstantially nontoxic mutein of Clostridium perfringens alpha-toxin ofthe present invention, (and/or an attenuated Clostridium perfringensorganism expressing the mutein) from those that are infected or had beeninfected by a wild type Clostridium perfringens alpha-toxin. The nextstep is to determine whether the antibody reacts with the fluid sample,e.g., binds to an antigen contained by the fluid sample. The animal isidentified as one that has/had been naturally infected by a Clostridiumperfringens organism when the antibody reacts with the fluid sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a genomic map of the C. perfringens alpha-toxinencoding region. The locations of the two large (1182 base pair and 1746base pair) fragments, respectively, that were used to construct CPERF001are shown. The location of the resulting 27 base pair deletion is alsoindicated. “Yplc” indicates the yplc gene (CPE0035); “plc” indicates thegene encoding alpha-toxin (a phospholipase C), and “CobW” indicates adownstream gene.

FIG. 2A illustrates the sequence of a portion of the plc gene from C.perfringens strain CP6 [SEQ ID NO: 4; this is a portion of SEQ ID NO:22, (i.e., nucleotides 262-300) of a plc gene fragment from CP6] and thecorresponding peptide sequence (SEQ ID NO: 5), where the underliningindicates the BamHI endonuclease restriction site used in creating thedeletion.

FIG. 2B illustrates the sequence of the primer (SEQ ID NO: 6) used tocreate the deletion in the parent C. perfringens strain 1240, givingrise to the deletant CPERF001. The underlining indicates the BamHlrestriction site included in the primer to facilitate construction ofthe deletion. Also illustrated is the corresponding peptide (SEQ ID NO:7).

FIG. 2C illustrates the sequence of the resulting deletion in CPERF001(SEQ ID NO: 8; nucleotides 103-117) and in the corresponding peptide(SEQ ID NO: 9). The underlining indicates the restored BamHlendonuclease restriction site.

FIG. 3A once again illustrates the sequence of a portion of the plc genefrom C. perfringens strain CP6 [SEQ ID NO: 4, nucleotides 262-300] andthe corresponding peptide sequence (SEQ ID NO: 5), where the underliningindicates the BamHl endonuclease restriction site used in creating thedeletion.

FIG. 3B illustrates the sequence of the primer (SEQ ID NO:10) used tocreate the deletion in the parent C. perfringens strain 29, giving riseto the deletant CPERF002. Also illustrated is the corresponding peptide(SEQ ID NO:11).

FIG. 3C illustrates the sequence of the resulting deletion in CPERF002(SEQ ID NO: 12) and corresponding peptide (SEQ ID NO: 13).

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the invention provides modified Clostridia organisms andcultures that express one or more of the Clostridia toxins, e.g.,alpha-toxins, as muteins that have no detectable toxicity, and/orsubstantially low toxicity, relative to the native or wild-typeClostridia toxins. Advantageously, the inventive C. perfringens mutantorganisms are readily administered as a live vaccine to animals. Theinventive mutein C. perfringens alpha-toxins are also provided, togetherwith nucleic acid molecules that encode the muteins, vectors forexpressing the alpha-toxins, and methods of using the same.

In order to provide a clear description of the invention, several termsare defined, as follows. A vaccine is a composition that includes animmunogen, and other optional pharmaceutically acceptable ingredients,including, in certain embodiments, suitable adjuvants. As used herein,the term “immunogen” describes a composition, substance or vector, thatwhen introduced into an animal, stimulates an immune response. Forpurposes of the present invention, an immunogen is contemplated toinclude any vector capable of expressing or introducing the inventivemutein alpha-toxin into an animal to be immunized. A vector includes,e.g., the inventive C. perfringens or other suitable microorganism, thatexpresses the inventive mutein alpha-toxin when the vector is introducedinto an animal. A vector also includes art-known nucleic acid molecules,e.g., plasmids and the like, that express the inventive muteinalpha-toxin when directly introduced into an animal, e.g, by entering acell of the animal and expressing the mutein alpha-toxin in the animal.An immunogen is also a protein, such as the inventive alpha-toxin,employed by itself or as part of a suitable vaccine composition.

As used herein, and unless otherwise specified, the terms “immunize” and“vaccinate” are synonymous and are used interchangeably to describe theintroduction of an immunogen into an animal to elicit an immune responsein the animal. The elicited immune response provides protective immunityto the treated animal that limits or reduces clinical disease signs,e.g., gas gangrene, and/or mortality, in vaccinated animals that arelater challenged with a virulent dose of a C. perfringens species forwhich the inventive vaccine is protective.

The term “adjuvant” is defined as one or more substances that causestimulation of the immune system. In this context, an adjuvant is usedto enhance an immune response to one or more vaccine antigens/isolates.An adjuvant may be administered to the target animal before, incombination with, or after the administration of the vaccine. Adjuvantsof the present invention may be obtained from any of a number of sourcesincluding from natural sources, recombinant sources, and/or bechemically synthesized, etc. Examples of chemical compounds used asadjuvants include, but are not limited to aluminum compounds;metabolizable and non-metabolizable oils; block polymers; ISCOM's(immune stimulating complexes); vitamins and minerals (including but notlimited to: vitamin E, vitamin A, selenium, and vitamin B12); Quil A(saponins); crosslinked acrylic acid-based polymers (e.g., prop-2-enoicacid polymers) crosslinked to different levels with a polyalkenylpolyether, as sold under the trademark CARBOPOL®; and/or uniformlydispersed micron size oil droplets in water emulsion, e.g., as soldunder the trademark Emulsigen®.

Additional examples of adjuvants, that sometimes have been referred tospecifically as immune stimulants, include, bacterial and fungal cellwall components (e.g., lipopolysaccarides, lipoproteins, glycoproteins,muramylpeptides, beta-1,3/1,6-glucans), various complex carbohydratesderived from plants (e.g., glycans, acemannan), various proteins andpeptides derived from animals (e.g., hormones, cytokines, co-stimulatoryfactors), and novel nucleic acids derived from viruses and other sources(e.g., double stranded RNA, CpG). In addition, any number ofcombinations of the aforementioned substances may provide an adjuvanteffect, and therefore, can form an adjuvant of the present invention.

The term “antibody” as used herein is intended to encompass polyclonalantibodies, monoclonal antibodies, and/or fragments or recombinantderivatives thereof, including engineered binding proteins incorporatingantibody variable domains.

As used herein, the residue numbering and position of the amino acidresidues of the inventive alpha-toxin proteins are based on the numbersystem described for Clostridium perfringens strain 13. The C.perfringens strain 13 alpha-toxin is reported by GenBank Accession No.NC 003366 as illustrated by SEQ ID NO: 1. The entire protein is 398amino acids long. The alpha-toxin encoded by the mutein vectorsexemplified hereinbelow correspond to the protein of SEQ ID NO: 1 havinga deletion of amino acid residues 90-98. There is a 28 amino acid signalsequence which is cleaved off during the maturation of the protein.Therefore the exemplified deletion corresponds to amino acid residues62-70 of the mature protein, that is 370 amino acids in length (SEQ IDNO: 3).

The codon numbering of the DNA encoding the inventive alpha-toxinproteins is based upon the plc gene of Clostridium perfringens strain13, as reported in GenBank Accession No. NP 560952, and as illustratedby SEQ ID NO: 2. The coding sequence for alpha-toxin runs fromnucleotide 48590 through 49786 in C. perfringens strain 13. The codondeletion in the two constructs exemplified herein corresponds tonucleotides 48857 through (and including) 48883 of the NP 560952 gene.This deletion is found within the alpha-toxin gene and corresponds tonucleotides 268-294 in the coding sequence of SEQ ID NO: 2.

Further, the use of singular terms for convenience in the description isin no way intended to be so limiting. Thus, for example, reference to acomposition comprising a C. perfringens cell includes reference to oneor more of such cells. It is also to be understood that this inventionis not limited to the particular configurations, process steps, andmaterials disclosed herein as such configurations, process steps, andmaterials may vary somewhat. It is also to be understood that theterminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims and equivalents thereof.

In a particular aspect of the present invention, non-reverting mutantsof C. perfringens are provided that are “substantially nontoxic,” i.e.,organisms that express an immunogenic alpha-toxin of little or notoxicity thereby rendering them suitable for use as protective vaccines.The inventive C. perfringens organisms therefore bestow a sufficientlyreduced toxicity, relative to wild-type C. perfringens organisms, torender them tolerable as a vaccine or antigen, when employed underconditions effective to elicit an anti-alpha-toxin oranti-C-perfringens, immune response in an animal that is so vaccinated.Thus, the inventive C. perfringens organism is “attenuated” relative tothe wild-type C. perfringens.

The phrase, “substantially nontoxic” is also intended to apply to theabove-noted immunogenic alpha-toxin muteins that have sufficiently lowor no toxicity, thereby also making them suitable for use in aprotective vaccine.

The reduction in toxicity is measured, e.g., by one of the followingart-known tests: hemolytic activity, phospholipase C activity,sphingomyelinase activity, phosphodiesterase activity, and generallethal activity in a test animal group. Generally, no residual toxicityis detectable by such standard tests. Nevertheless, the presence of aminimal level of one or more of these activities, e.g., from about 10⁻⁴to about 10⁻², relative to the toxicity of an equivalent number ofinfectious units of the wild-type C. perfringens alpha-toxin from whichthe mutein has been derived, may prove acceptable in a veterinarysetting.

In a one embodiment, the invention is practiced employing biotype Astrains of C. perfringens, which are of particular importance as theetiological agents of various types of gangrene and enteric diseases. Inparticular, the alpha-toxin of C. perfringens is the target fordeletion-attenuation, since attenuation of this toxin is sufficient torender C. perfringens sufficiently non-lethal, relative to wild-typestrains.

Broadly, the inventive C. perfringens plc gene expresses a muteinalpha-toxin. The mutein alpha-toxin has a deletion that includes the Hisof the Zn2 loop, together with flanking residues, in order to greatlyreduce the possibility of any back mutation to the toxic form. It hasnow been found that deletion of the Zn2 loop His residue, e.g., His₆₈ ofSEQ ID NO: 3, together with the deletion of additional residues flankingthe His residue of the Zn2 loop, provides an alpha-toxin retainingsufficient immunogenicity to induce protective immunity in animalsvaccinated with the inventive C. perfringens, while also being unlikelyto undergo a back-mutation to encoding a wild-type alpha-toxin. Theadditional residues that can be deleted are deleted in the C-terminaldirection and/or in the N-terminal direction, relative to His₆₈, and canrange in number from about 4 through about 60 residues in either ofthose directions. Alternatively, His₁₄₈ and flanking residues, can besimilarly deleted.

One embodiment of the inventive mutein alpha-toxin also includes, inaddition to the deletion of His₆₈, a deletion of at least 30 amino acidresidues from either side (in the C-terminal or N-terminal direction) ofthe His₆₈ position, relative to SEQ ID NO: 3. In another embodiment, theinventive mutein alpha-toxin includes, in addition to the deletion ofHis₆₈, a deletion of at least 20 amino acid residues from either side ofHis₆₈, relative to SEQ ID NO: 3. In still another alternativeembodiment, the inventive mutein alpha-toxin includes, in addition tothe deletion of His₆₈, a deletion of at least 5 amino acid residues fromeither side of His₆₈, relative to SEQ ID NO: 3. In yet anotherembodiment, the inventive mutein alpha-toxin includes a deletion fromabout residue 62 through about residue 70, relative to SEQ ID NO: 3.Optionally, the deleted amino acid residues are replaced by one or moreother residues, such as a single leucine residue.

In still further another embodiment, the mutein C. perfringensalpha-toxin is produced and isolated from C. perfringens, or from analternative recombinant organism, to be employed as a research reagent,and/or in diagnostic kits or assays, e.g., as a target foranti-alpha-toxin antibodies. Yet another utility for the mutein C.perfringens alpha-toxin proteins is in specialized vaccines for animals,e.g., humans, that may not tolerate vaccination with the inventiveattenuated C. perfringens organism.

In addition, the present invention provides an antibody thatspecifically binds to the wild-type alpha-toxin relative to thealpha-toxin muteins of the present invention.

In a further embodiment, an antibody is provided that preferentiallybinds to wild-type C. perfringens alpha-toxin protein while exhibitingminimal or no binding to the inventive mutein alpha-toxin, e.g.,avoiding binding to an alpha-toxin mutein that has a deletion asdescribed in detail, supra. The antibody provided is therefore usefulfor distinguishing the deletion mutein alpha-toxin from the wild-typealpha-toxin, and thereby, also useful for distinguishing animalsvaccinated with a vaccine of the present invention from animals thathave been infected by wild type C. perfringens. Methods of eliciting andscreening for such selective antibodies are art-known. Similarly,antibodies that recognize the inventive mutein alpha-toxin of thepresent invention, but not the wild-type protein can also be generated.The antibodies of the present invention can be polyclonal, monoclonal(“mAb”) or fragments or engineered fragments or derivatives of suchantibodies retaining selective binding properties.

Techniques for preparing and screening monoclonal antibodies have beenamply described [see, e.g., Stites, et al. (eds.) Basic and ClinicalImmunology (4th ed.), Lange Medical Publications, Los Altos, Calif.(1988); Harlow and Lane, Antibodies: A Laboratory Manual, CSH Press(1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed.),Academic Press, New York (1986); and Kohler and Milstein, Nature256:495-497 (1975), all of which are incorporated by reference herein intheir entireties].

For instance, and without limitation, an immune response is elicited insuitable animals, such as a mouse or chicken, by vaccination with apurified wild-type C. perfringens alpha-protein, e.g., in combinationwith a suitable adjuvant. For example, in order to avoid the toxicity ofthe wild-type alpha protein, the immunogen is a peptide corresponding tothe deleted residues, and, if necessary the immunogenicity of thepeptide is enhanced by combination with a suitable adjuvant or bycoupling to a suitable carrier protein. Coupling to a carrier protein isart known, and can be accomplished, e.g., by providing the peptide witha terminal cysteine, and coupling it to keyhole limpet hemocyanin (KLH)or bovine serum albumin (BSA) using either maleimide coupling chemistryor sulfosuccinimidyl 4-[N maleimidomethyl]cyclohexane-1-carboxylate)linker (from Pierce) A carbodiimide linker also may be employed withoutrequiring a terminal cysteine.

For preparation of monoclonal antibodies, splenic lymphocytes can beobtained from an immunized animal, hybridomas are prepared from thoselymphocytes, and one or more potentially suitable hybridomas expressinganti-alpha protein can be obtained. The hybridomas are screened againstboth mutein and wild-type alpha-protein, and a hybridoma expressing anantibody that binds only to the wild-type alpha-toxin is identified,cloned and employed to produce monoclonal antibodies that bind only tothe wild-type alpha protein. Optionally, cDNA from the identifiedhybridoma clonal line is obtained, and recombinant antibodies orantibody fragments can be produced in other art-known expressionsystems.

As discussed above, one potential disadvantage of vaccinations ingeneral is that the resulting vaccinated animals can generate falsepositives when testing for infection using antibodies raised against anaturally occurring strain, thereby, hindering the identification ofinfected animals. Therefore, the present invention provides a test kitfor distinguishing a subject animal that has been infected by anaturally occurring C. perfringens organism from one vaccinated with amutein alpha-toxin of the present invention.

One such test kit includes a quantity of a selective anti-wild-typealpha-toxin antibody that exhibits minimal or no binding to a muteinalpha-toxin of the present invention. The kit can also include othersuitable reagents, sufficient for conducting at least one diagnostictest. In a further embodiment, the antibody is tagged or labeled with areadily detectable maker moiety, e.g., any art-known enzymatic marker,e.g., peroxidase; fluorescent tag, e.g., fluorescein; beads, includingmagnetic beads; and the like. Optionally, the kit can further include atagged antibody that selectively binds to the selective anti-wild-typealpha-toxin antibody. Immunoassays are well known to the art, andinclude sandwich immunoassay, competitive immunoassays, enzyme-linkedimmunosorbent assays (ELISA), radioimmunoassays (RIA) and others.

Also provided are methods for identifying and distinguishing an animalthat has been infected by a naturally occurring, i.e., wild-type, C.perfringens organism, from an animal vaccinated with a vaccinecomprising the attenuated C. perfringens organism of the invention, thatis conducted, for example, by the following steps:

(a) contacting a fluid sample from the animal with an above-describedselective anti-wild-type alpha-toxin antibody that exhibits minimal orno binding to a mutein alpha-toxin of the present invention; and

(b) determining whether the antibody reacts with the fluid sample;wherein when the antibody reacts with the fluid sample, the animal isidentified as one that has been infected by a naturally occurring C.perfringens organism.

Producing Attenuated C. Perfringens Strains

A process of converting a wild-type C. perfringens isolate into anattenuated, or substantially nontoxic, strain suitable to beadministered as a vaccine can be conducted as follows. The alpha-toxin(plc) gene can be replaced on the bacterial chromosome with a geneencoding only an alpha-toxin mutein, leaving no remaining capability forthe C. perfringens organism to produce wild-type alpha-toxin. Verybroadly, the process of producing the vaccine organism includes, but isnot limited to, the following general steps, not necessarily in theorder presented.

-   -   (1) Identifying the type of animal to be protected by        vaccination and one or more clinical isolates obtained for        screening purposes. This step is typically optional in the case        for the alpha-toxin, since isolates from one species of animal        are more likely than not to provide protection for other species        of animal.    -   (2) Amplifying the plc gene from the C. perfringens isolate or        isolates, e.g., by PCR or other art-known nucleic acid        amplification technique, with suitable flanking primers, and        employing amplification with suitable primers to create the        desired deletion mutation. Alternatively, an appropriate library        can be probed.    -   (3) Creating a suicide vector comprising the deletion plc gene,        in which the C. perfringens origin of replication has been        removed, and/or an origin of replication that can replicate        in C. perfringens is simply not present; and in either case,        including suitable selectable markers, e.g., antibiotic markers,        adjacent to the mutated plc gene. Such a vector can then be        inserted into C. perfringens organisms, e.g., by        electroporation, or other art-known methods.    -   (4) Selecting C. perfringens organisms in which the mutein plc        gene has been successfully integrated into the bacterial        chromosome. This is done by culturing the C. perfringens        organisms of step (3) in the presence of the selectable agent,        e.g., an antibiotic(s) corresponding to the selectable markers.        For example, the only C. perfringens organisms that would grow        under antibiotic selection would be those that through        homologous recombination have directly integrated the suicide        vector, with its antibiotic resistance gene(s), into the        bacterial chromosome. These growing C. perfringens organisms        therefore, would have two adjacent plc genes, one being        wild-type, whereas the other would have the deletion mutation.    -   (5) Selecting C. perfringens organisms that have undergone a        further recombination event that removes the selectable markers,        e.g., antibiotic markers, along with the wild-type plc gene.        This is done by culturing the organisms of (4) in the absence of        the selectable agent, e.g., the antibiotic, and selecting for        non-hemolytic clones on blood agar.

Since the insertion of the mutein nucleic acid is accomplished byhomologous recombination, the nucleic acid molecule encoding the muteinalpha-toxin is incorporated at a chromosomal position that is homologousto the location of a nucleic acid molecule encoding a wild-typealpha-toxin that is present in non-attenuated Clostridium perfringens.

In more detail, field isolates of C. perfringens are obtained fromdiseased animals or other sources. Initially, genomic DNA obtained fromfield isolates of interest is inserted into a suitable dualmicroorganism shuttle vector, e.g., a shuttle plasmid with selectablemarkers, e.g., antibiotic markers, to assess their transformability.Broadly, a suitable shuttle vector will include one, two, three or moreof the following features, a cloning site, a C. perfringens origin ofreplication, an E. Coli origin of replication, and an antibioticresistance gene andor selectable marker. Art-known vectors suitable forthis purpose, or readily adaptable for this purpose include, forexample, the recombinant shuttle plasmid pHR106 described by Roberts etal., [Appl Env Mircobiol 54: 268-270 (1988)]; the PJIR 750 and PJIR 751plasmids described by Bannam, et al., [Plasmid 29:233-235 (1993)]; thepromoterless PPSV promoter selection vector of Matsushita, et al., 1994,Plasmid 31, 317-319; the shuttle plasmids pJIR1456 and pJIR1457,described by Lyras, et al., 1988, Plasmid 39, 160-164; and the pAK201shuttle vector described by Kim et al., 1989, Appl Environ Microbiol 55,360-365, the contents of which are incorporated herein by reference intheir entireties. Removal of the C. perfringens origin of replicationconverts the shuttle vector into a suicide vector.

For example, one shuttle plasmid is pJIR418, described by Sloan, et al.,1992, Plasmid 27, 207-219, incorporated by reference herein.

Isolates yielding >104 transformants per microgram of plasmid DNA, andthat are susceptible to the antibiotic marker, e.g., chloramphenicol orerythromycin, are potential candidates for deletion. Genomic DNA fromthe candidate strains is then used as a template for long range PCR ofthe plc (alpha-toxin) gene and flanking sequences of the candidatestrains. For example, as exemplified hereinbelow, the C. perfringensstrain 13 chromosome was used to identify primers for amplifying thegene encoding the alpha-toxin. These primers were then used to clone thealpha-toxin gene from another strain, that was the CP6 poultry isolate.

After subcloning of the PCR products, the alpha-toxin gene and flankingregions are sequenced and restriction mapped. New oligonucleotideprimers are synthesized with flanking restriction sites and the productsof two separate amplifications are cloned into a suitable suicideplasmid (C. perfringens origin of replication has been removed), e.g.,exemplified hereinbelow as plasmid 1192-23.1 to create the desiredvaccine strain with the deletion.

The provided suicide vector(s) specific to the isolate(s) are insertedinto the corresponding animal strain of C. perfringens, by any standardart-known method. For example, this is accomplished by electroporation.When the suicide vector is inserted into C. perfringens (without the C.perfringens origin of replication, it is unable to replicate in thecytoplasm, and does not survive unless it successfully integrates intothe bacterial chromosome.) Successful integrants are the only organismsthat will grow in the presence of the antibiotic corresponding to thenewly introduced antibiotic marker gene.

Any art-known selectable marker gene may be employed, althoughchoramphenicol and/or erythromycin markers are employed in the vectorsexemplified hereinbelow.

These recombinant events result from homology of the wild-type plc geneto the deletion plc gene plasmid DNA. The resulting recombinant bacteriais termed an integrant. The integrant contains a copy of the introducedhomology vector that is integrated at the plc gene locus. Thus, theresulting integrant includes two copies of the plc gene, the originalnormal copy, and the introduced deleted version. The introducedantibiotic resistance genes are located between the two copies of theplc gene. Rare random recombination events can occur between the twocopies of the plc gene. This recombination event can produce one of twooutcomes. In both cases, the DNA intervening between the two copies ofthe plc gene (including the resistance genes) has been removed. In thefirst outcome, the normal or wild-type plc gene is restored, resultingin recovery of the original parent strain without antibiotic markers. Inthe second outcome, the wild-type plc gene is replaced by the deletedcopy, producing the desired alpha-toxin deletant construct, without theantibiotic markers.

Removing the antibiotic from the culture medium allows those bacteriathat have undergone recombination to survive and replicate. The deletionrecombinant clones are then identified by growth on blood agar, withoutthe hemolysis normally exhibited by C. perfringens that expresses thewild-type alpha-toxin.

Animals to be Vaccinated

Animals for which an attenuated C. perfringens vaccine can be produced,and from which a useful C. perfringens wild-type strain may be isolated,include, broadly, any animals for which C. perfringens infection is aproblem. Vertebrates of interest include avians, mammals, and fish, andparticularly animals of economic and/or agricultural importance. Thefollowing list of animals are those that are contemplated to benefitfrom a C. perfringens vaccine and/or from which useful C. perfringenswild-type isolates may be obtained. While it is sometimes possible thatany such vaccine comprise a component (living or non-living) that wasoriginally isolated from the same genus or species of animal that is tobe vaccinated, this is not a requirement.

A non-limiting list of such animals include those of the avian, bovine,ovine, etc., families, as well as aquatic animals, e.g., that may besubjected to aquaculture and/or harvested from the wild and kept alivein holding tanks for a time prior to marketing. These include fish suchas trout or salmon, and other species raised or harvested for economicbenefit. Non-vertebrate aquatic animals include lobsters, crabs,mollusks, e.g., squid, octopus, clams, oysters, muscles, scallops, andthe like. Avian shall be understood to include, for example, chickens,turkeys, geese, duck, etc. Bovine shall be understood to include, forexample, cattle, beef, veal, etc. Ovine shall be understood to include,for example, lamb, etc.

For purposes of the present invention, the term “fish” shall beunderstood to include without limitation, the Teleosti grouping of fish,i.e., teleosts. Both the Salmoniformes order (which includes theSalmonidae family) and the Perciformes order (which includes theCentrarchidae family) are contained within the Teleosti grouping.

Examples of potential fish recipients include the Salmonidae family, theSerranidae family, the Sparidae family, the Cichlidae family, theCentrarchidae family, the three-Line Grunt (Parapristipoma trilineatum),and the Blue-Eyed Plecostomus (Plecostomus spp). TAXON NAME COMMON NAMESalmonidae Family Coregonus clupeaformis Lake whitefish Coregonus hoyiBloater Oncorhynchus keta Chum salmon Oncorhynchus gorbuscha Pink salmonOncorhynchus kisutch Coho salmon (silver salmon) Oncorhynchus masoucherry salmon (masou salmon) Oncorhynchus nerka Sockeye salmonOncorhynchus tshawytscha (chinook salmon) Prosopium cylindraceum Roundwhitefish Oncorhynchus clarki Cutthroat trout Oncorhynchus mykissRainbow trout Salmo salar Atlantic salmon Salmo trutta Brown trout Salmotrutta X S. fontinalis Tiger hybrid-trout Salvelinus alpinus Arcticcharr Salvelinus confluentus Bull trout Salvelinus fontinalis Brooktrout Salvelinus leucomaenis Japanese charr (white spotted charr)Salvelinus malma Dolly varden (Miyabe charr) Salvelinus namaycush Laketrout Thymallus thymallus Grayling Some Members of the Serranidae FamilyCentropristis ocyurus Bank sea bass Centropristis philadelphicus Rocksea bass Centropristis striata Black sea bass Diplectrum bivittatumDwarf sandperch Diplectrum formosum Sand perch Epinephelus flavolimbatusYellowedge grouper Epinephelus morio Red grouper Serranus phoebe TattlerSerranus tortugarum Chalk bass Some Members of the Sparidae familyArchosargus probatocephalus Sheepshead Archosarpus rhomboidalis Seabream Calamus penna Sheepshead porgy Lagodon rhomboides Pinfish PagrusMajor Red Sea bream Sparus aurata Gilthead Sea bream Stenotomus chrysopsScup Some Members of the Cichlidae family Aepuidens latifrons Blue acaraCichilsoma nigrofasciatum Congo cichlid Crenichichla sp. Pike cichlidPterophyllum scalare Angel fish Tilapia mossambica Mozambique mouthbreeder Oreochromis spp Tilapia Sarotherodon aurea Golden Tilapia SomeMembers of the Centrarchidae family Ambloplites rupestris Rock bassCentrarchus macropterus Flier Elassoma evergladei Everglades pigmysunfish Elassoma okefenokee Okefenokee pigmy sunfish Elassoma zonatumBanded pigmy sunfish Enneacanthus gloriosus Bluespotted sunfishEnneacanthus obesus Banded sunfish Lepomis auritus Red breast sunfishLenomis cyanellus Green sunfish Lepomis cyanellus X L. gibbosus Green xpumpkinseed Lepomis gibbosus Pumpkinseed Lepomis gulosus WarmouthLepomis humilis Orange-spotted sunfish Lepomis macrochirus BluegillLepomis megalotis Longear sunfish Micropterus coosae Shoal bassMicropterus dolomieui Small mouth bass Micropterus punctulatus Spottedbass Micropterus salmoides Largemouth bass Pomoxis annularis Whitecrappie Pomoxis nigromaculatus Black crappie

In a further embodiment, the animal is a companion animal or a human.For purposes of the present invention, the term “companion” animal shallbe understood to include all animals—horses (equine), cats (feline),dogs (canine), and rodents, including mice, rats, guinea pigs, rabbitspecies, and avians, such as pigeons, parrots, and the like.

Birds receiving such vaccination can be associated with eithercommercial or noncommercial aviculture. These include e.g., Anatidae,such as swans, geese, and ducks, Columbidae, e.g., doves and pigeons,such as domestic pigeons, Phasianidae, e.g., partridge, grouse andturkeys, Thesienidae, e.g, domestic chickens, Psittacines, e.g.,parakeets, macaws, and parrots, e.g., raised for the pet or collectormarket. Chickens are exemplified hereinbelow.

Sources of Wild-Type Isolates

Generally, the attenuated C. perfringens organisms of the invention canbe produced starting with a wild-type C. perfringens that has originallybeen isolated from any infected animal of interest, as discussed supra,and/or from the environment. The environment includes any material thatcontains viable C. perfringens organisms and/or viable C. perfringensspores including, for example, contaminated food, soil, water, animalbedding material, feces, and the like.

Justin et al., [Biochemistry 41, 6253-6262 (2002)] characterizedalpha-toxins from different strains of C. perfringens that are almostidentical in sequence and biochemical properties. However, Justin etal., also describe a strain that was isolated from an avian source (aswan) that had an alpha-toxin exhibiting a large degree of sequencevariation and altered substrate specificity compared to the otherstrains. For this reason, it is believed that most isolates will, whenconverted to an attenuated form, elicit protective immunity against thealpha-toxin of many other naturally occurring strains of C. perfringens.Nevertheless, given the possibility of alpha-toxin variation betweenisolates, it will often be advantageous to isolate and attenuate C.perfringens organisms from the animal species for which an anti-C.perfringens vaccine is desired. The isolate exemplified hereinbelow wasisolated from chicken, and tested in that species.

Vaccines

The attenuated C. perfringens organisms of the invention are generallyformulated into pharmaceutically acceptable vaccine compositions. Thevaccine compositions are formulated according to the route ofadministration and are compatible with the active antigenic agent. Theactive antigenic agent is, for example, one or more strains ofattenuated C. perfringens type A organisms according to the invention.Optionally, the vaccine composition can also include one or more typesof non-toxic alpha-protein, in combination with the attenuated C.perfringens type A organisms.

For example, substantially all of the attenuated C. perfringens type Aorganisms included in the vaccine composition are alive and viable,although for certain specific situations, e.g., for immunizing certainhumans or immune-compromised animals, the vaccine will exclusivelycomprise killed attenuated C. perfringens type A organisms.

The vaccine composition includes physiologically compatible buffersand/or salts, in optional combination with adjuvants and/or optionalimmune enhancers or stimulants (co-administered or administered inseries, e.g., before or after vaccination).

Suitable immune stimulants include, but are not limited to, cytokines,growth factors, chemokines, supernatants from cell cultures oflymphocytes, monocytes, cells from lymphoid organs, cell preparations orcell extracts (e.g. Staphylococcus aureus or lipopolysaccharidepreparations), mitogens, or adjuvants including low molecular weightpharmaceuticals. An immune stimulant can be administered in ovo at anytime during incubation. In a particular aspect, the immune stimulant isadministered in the medium containing the attenuated C. perfringens typeA organisms.

Methods of Administering Vaccine

The above-described inventive vaccines are administered, for example, byinjection or inoculation by one or a combination of the followingroutes: oral, intranasal, parenteral, subcutaneous, scarification,and/on intramuscular administration in any suitable, art-knownformulation, e.g., a compatible buffer and/or a physiologicallyacceptable saline, in optional combination with adjuvants and/or immuneenhancers or stimulants (co-administered or administered in series,e.g., before or after vaccination). For orally-administeredvaccines/vaccination methods, any of the physiologically-suitablebuffers or suspending agents that are known to the art are readilyemployed. In addition, the composition can be incorporated, e.g.,admixed into drinking water or sprayed onto food pellets, dusted orsprayed onto corn or other grains, and the like.

The gastrointestinal tract is a common site of C. perfringens infection,and therefore oral administration is contemplated as one method ofinoculation. The presence of the gastrointestinal tract by the liveattenuated C. perfringens organisms of the invention is contemplated toelicit localized protective immune reactions in the mucosal layer of thegastrointestinal tract, and may also act competitively to preventsubsequent colonization by wild-type C. perfringens.

For avians, such as domestic fowl, including chickens, ducks, geese,etc., the oral method, or injection in ovo are among the useful routesfor vaccination. The in ovo route is exemplified hereinbelow, andproduced active immunization and protection from challenge in thehatched chickens.

Foreign Gene Expression by C. Perfringens

Using the techniques developed in the preceding examples, any gene notnaturally occurring, i.e., foreign to C. Perfringens optionally can beinserted into the chromosomal DNA of C. perfringens. For expression offoreign proteins, gene fusions can be made which preserve the sequencesflanking the alpha-toxin gene, the alpha-toxin promoter and its signalsequence. In one embodiment, most of the coding sequence of the plc geneis replaced with the foreign gene. The remaining nucleotides of the plcgene downstream of the inserted gene are out of frame, and therefore nofunctional alpha-toxin is produced. Alternatively, the foreign gene canbe inserted in-frame to the nucleotide sequence encoding the alpha-toxinmutein forming an alpha-toxin—foreign protein fusion protein.

Oligonucleotide primers for the foreign gene can be synthesized, e.g.,with an N-terminal FLAG tag sequence and appropriate restriction sites.The PCR products can be cloned into the suicide vector; the FLAG tag andforeign gene are inserted in frame with the alpha-toxin signal sequence.The foreign protein is expressed under control of the alpha-toxinpromoter and targeted for secretion by the plc signal sequence. Thesecreted foreign protein can be detected in the supernatant media byWestern blot using an anti-FLAG antibody.

Any suitable foreign gene may be inserted into the C. perfringens genomein this manner. These include, for example, DNA encoding antigens frompathogens of the gastrointestinal tract, including, e.g., antigenicproteins of bacteria such as E. coli, salmonella species, campylobacterspecies, lawsonia species, and the like; antigenic proteins of parasitessuch as eimeria species, isospora species, cryptosporidium species andthe like; and antigenic proteins of viruses such as rotaviruses,coronaviruses, and the like, in order to immunize the animal treatedwith such a recombinant C. perfringens. Other proteins that may beexpressed by such recombinant C. perfringens include therapeuticproteins or peptides. Optionally, these include peptides that areendogenous to the gastrointestinal tract, including trefoil factor, orany type of art-known cytokine, e.g. chicken IL-18, and the like.

One such C. perfringens construct expresses the chicken IL-18 protein.Administration of live bacteria containing the gene fusion will allowdelivery of therapeutic doses of IL-1 8 into the gut yet be relativelyinnocuous to the host animal by virtue of the absence of alpha-toxinproduction. Other therapeutic agents may also be expressed using thissystem.

Numerous references are cited herein, the content of each of which isincorporated by reference herein in its entirety. The following specificexamples are included for purposes of illustration, and are not intendedto limit the scope of the invention, unless otherwise indicated.

EXAMPLE 1 C. perfringens alpha-toxin Deletant Homology Vector

A homology plasmid vector 1162-55-20 useful in the construction of C.perfringens alpha-toxin deletants was created. The plasmid incorporatesseveral important elements; the replication region from the E. coliplasmid pUC1 8; the C. perfringens chloramphenicol (catP) anderythromycin (ermBP) resistance genes (both of which are also expressedin E. coli); and a C. perfringens alpha-toxin gene (plc) inactivated bya specific deletion of 9 amino acids. Plasmid 1162-55-20 was created inseveral steps, as follows.

First the plc gene was cloned from a recent avian isolate of C.perfringens (strain CP6). The sequence of C. perfringens strain 13(Genbank NC 003366; SEQ ID NO: 2) was used to design oligonucleotideprimers to be used in the cloning of the plc gene. These and allsubsequent primers were obtained commercially form Sigma Genosys,Woodlands, Tex. The upstream primer located within the yplc gene(CPE0035), 5′ AGCTGCATAAGCAAAAGTTCCAACTC 3′ (SEQ ID NO: 14) correspondsto nucleotides 47675-47700 of strain 13 (SEQ ID NO: 2). The downstreamprimer located within the cobwgene (CPE0037), 5′GCAGAAACTCTTCTTAGACCTATTCTTTTAGGC 3′ (SEQ ID NO: 15), is complementaryto nucleotides 50597-50629 of strain 13. These primers were used alongwith genomic DNA from C. perfringens strain CP6 in a long rangepolymerase chain reaction (PCR). A product of 2955 base pairs from theupstream yplc gene through the downstream cob W gene was predicted fromthe known sequence of strain 13 (as illustrated by FIG. 1). Thealpha-toxin promoter, signal sequence and plc gene (CPE0036) codingsequence are contained within this fragment, i.e. between the upstreamyplc gene and the downstream cob W gene. The PCR 2955 base pair fragmentwas then cloned into cloning vector pCR-Blunt (Invitrogen Corporation,Carlsbad, Calif.) resulting in plasmid 1162-52.1. The nucleotidesequence of the plc coding region of the 2955 base pair fragment ofstrain CP 6, was determined from this fragment (SEQ ID NO: 22; andcorresponding polypeptide is SEQ ID NO: 23) and was shown to besubstantially homologous as that of the plc gene of C. perfringensstrain 13 (SEQ ID NO: 2).

Next the E. coli replication region and the C. perfringens resistancegenes were cloned from shuttle plasmid pJIR418 (Sloan, et al, 1992,Plasmid 27, 207-219; Genebank M77169). Plasmid pJIR418 was digested withrestriction enzymes Bam HI and Spe I and the ends filled in with Klenowpolymerase. Ligation of the large fragment produced plasmid 1162-45.1and restored the Bam HI restriction site. This plasmid retains the E.coli replication region, but unlike pJIR418, it does not contain a C.perfringens origin of replication. The plasmid is therefore capable ofautonomous replication in E. coli, but not in C. perfringens.

In the next step the C-terminal half of the plc gene was sub-cloned intothe intermediate plasmid 1162-45.1. Digestion of plasmid 1162-52.1 withBam HI and Eco RI released a 1742 base pair fragment containing theC-terminal portion of the alpha-toxin gene from the unique Bam HI sitelocated with in the plc gene downstream through the cob W gene to an EcoRI site located in the multiple cloning site of the parent plasmid. The1742 base pair fragment was cloned between the Bam HI and Eco RI siteslocated within the multiple cloning site of plasmid 1162-45.1. In theresulting plasmid 1162-53.7, the C-terminal half of the plc gene is inthe same transcriptional orientation as the catP and ermBP genes of theparental plasmid.

In the final step the N-terminal half of the plc gene was cloned intothe unique Bam HI site of plasmid 1162-53.7. This was accomplished bycreating a PCR fragment derived from the alpha-toxin gene sub-cloned inplasmid 1162-52.1. The upstream primer, 5′ggatccAGCTGCATAAGCAAAAGTTCCAACTC 3′ (SEQ ID NO: 16) was identical to thepreviously noted yplc primer (SEQ ID NO: 4) except that a flanking BamHI site (lowercase) was included. The downstream primer located withinthe alpha-toxin gene, 5′ ggatccaATGCATTCTTATCATAATCTGGATAAGTAGAACC 3′(SEQ ID NO: 17) was complementary to nucleotides 48824-48857 of strain13 and included a flanking Bam HI restriction site and a spacernucleotide to maintain the reading frame (lowercase). The Bam HIfragment resulting from PCR with these primers and plasmid 1162-52-1template DNA was cloned into the unique Bam HI site of plasmid1162-53.7. A plasmid containing the two plc gene regions in the sametranscriptional orientation was isolated. This plasmid 1162-55.20contains the plc gene with the desired nine amino acid deletion and theaddition of a leu residue between the ala 61 and asp 71 of the wild-typetoxin (see FIG. 2). DNA sequencing of the plasmid confirmed the readingframe and the net deletion of 24 codons (encoding eight amino acidresidues).

EXAMPLE 2 Construction of Clostridium perfringens Recombinant CPERF001

The deleted version of the plc gene constructed in Example 1 wasintroduced into C. perfringens using the following strategy. Thehomology vector 1162-55.20 is termed a C. perfringens “suicide plasmid”because its C. perfringens origin of replication has been removed.

When this plasmid was transformed into C. perfringens it is unable toreplicate and does not survive. However, if the transformed bacteria areplaced under chormaphenicol and/or erythromycin selection, the plasmidDNA can be forced to recombine into the bacterial genome via homology tothe plc gene. The resulting recombinant bacteria is termed an integrant.The integrant contained a copy of the introduced homology vector thatwas the plc gene locus. Thus, the resulting integrant contained twocopies of the original normal copy, and the introduced deleted version.The introduced resistance genes were located between the two copies ofthe plc gene. When antibiotic selection was removed from the integrant,recombination occurred between the two copies of the plc gene. Thisrecombination event can produce one of two outcomes. In both cases, theDNA intervening between the two copies of the plc gene (including theresistance genes) has been removed. In the first outcome, the normal plcgene was restored, resulting in recovery of the original parent strain.In the second outcome, the normal plc gene is replaced by the deletedcopy, producing the desired alpha-toxin deletant construct. Since thealpha-toxin of the deletant strain is inactivated, this strain wasnon-hemolytic. Therefore the desired deletant integrant was identifiedby screening for non-hemolytic clones on blood agar plates.

Because the recombination event resulting in the desired integrant wasexpected to occur at a low frequency, it was critical to employ parentC. perfringens strains having high transformation efficiency. Thereforeseveral recent avian isolates of C. perfringens were analyzed fortransformation efficiency. The isolates were transformed with theshuttle plasmid pJIR418 as described by Allen and Blaschek (Applied andEnvironmental Microbiology 54:2322 1988)) with slight modifications(described below). Strain 1240 exhibited the highest transformationefficiency (see Table 1), 9.2×10⁶ transformants/μg of pJIR418 plasmidDNA. This strain was chosen to be the parent strain for construction ofdeletant CPERF001. Strain 29 was chosen to be the parent strain forCPERF002. TABLE 1 Transformation Efficiency of C. perfringens AvianIsolates C. perf. A Strain Transformants/μg of pJIR418  29 3.6 × 10⁴  235.6 × 10² 1220 4.0 × 10⁶ 1240 9.2 × 10⁶ CP-2 None 1230 7.1 × 10³ 5227None 5230 NoneSource of above listed wild-type strains was Dr. J. Glenn Songer, Dept.of Veterinary Sciences and Microbiology, University of Arizona, Tucson,Arizona 85721

C. perfringens strain 1240 cells from an overnight anaerobic culture inTSYC media (30 g/l tryptic soy broth, 5 g/l yeast extract, 0.5 g/lcysteine) were diluted 1:25 and grown to A₆₀₀=0.5436. Aftercentrifugation of 100 ml of cells at 18,000×g for 10 minutes,electrocompetent cells were prepared by twice resuspending cells in anequal volume of pre-reduced sucrose magnesium phosphate (“SMP” wasprepared as 270 mM sucrose, 1 mM MgCl₂, 7 mM NaPO₄, pH 7.3) bufferfollowed by a final resuspension with 0.5 ml SMP. This resulted in afinal volume of ˜2.0 ml.

Aliquots of 100 μl of cells were electroporated with 4 μg of plasmid1162-55.20 in 0.2 cm cuvettes. A Bio-Rad Gene Pulser II was used at 1.37kilovolts, 100 Ohms resistance and 50 microfarads capacitance.Immediately after electroporation, cells were diluted with 2.0 ml ofpre-reduced tryptic soy yeast cysteine media (“TSYC” was prepared as 30g tryptic soy broth, 5 g yeast extract, 0.5 g cysteine, 950 ml water)and incubated for three hours at 37° C. in an anaerobic jar. After therecovery period, dilutions of cells were plated on TSYC +25 μg/ml ofchloramphenicol plates. These plates were incubated at 37° C. overnightin an anaerobic jar.

After overnight growth, an average of 50 chloramphenicol-resistantcolonies per microgram of 1162-55.20 DNA was observed. Single coloniesof seven putative integrants were grown up in nonselective TSYC mediafor four successive grow outs and plated onto nonselective blood agarplates. One of the non-selected stocks, 1192-31.7, exhibited severalnon-hemolytic colonies. Twenty-one of these non-hemolytic colonies werepatched to nonselective master plates and replica plated onTSYC+chloramphenicol plates. Two of the 21 colonies were chloramphenicolsensitive.

One of the chloramphenicol-sensitive putative deletants, 1192-32.14, wasgrown up along with the 1240 wild-type and the integrant 1192-31.7 andplated on blood agar plates. The 1240 wild-type strain showed clearzones of beta hemolysis whereas the 1192-32.14 deletant was a pureculture of non-hemolytic colonies and was renamed CPERF001.

Other blood plates were used as masters and replica plated tononselective and selective media. The 1240 wild-type and CPERF001 werechloramphenicol and erythromycin sensitive. The integrant, 1192-31.7,was both chloramphenicol and erythromycin resistant as expected.

To rule out the possibility that the antibiotic resistance genes werepresent but not expressed in CPERF001, PCR primers specific for thechloramphenicol and erythromycin genes were synthesized for use in PCRreactions. Genomic DNAs were prepared from the wild-type, integrant andCPERF001 strains and used as templates for PCR reactions with theantibiotic gene primers. Results of the PCR analysis showed positiveresponse only from the suicide plasmid and the integrant and not theparent 1240 or deletant CPERF001. This confirms the predicted loss ofthe resistance gene sequences.

To confirm the deletion within the alpha-toxin gene, a 1086 bp regionsurrounding the deletion was amplified by PCR using appropriatealpha-toxin specific primers. The amplified fragment was cloned andsequenced. Sequencing results confirmed the deletion of the nine aminoacids from tyr 62 through trp 70 of the alpha-toxin gene and theinsertion of a single leu (see FIGS. 2A-2C).

CPERF001 was assayed for expression of the inactivated alpha toxoidprotein. After 6 hours of anaerobic growth, aliquots of 1 ml of cellswere collected and centrifuged. Fifteen microliters of theunconcentrated supernatant media were analyzed by polyacrylamide gelelectrophoresis and subjected to Western Blot analysis with a rabbitpolyclonal antibody directed against recombinant alpha-toxin protein(Vaccine 11(12): 1253-1258 (1993)). The results showed specific antibodyreactivity with a protein of the expected size for the alpha toxoidprotein.

CPERF001 is a genetically engineered deletant strain of C. perfringensType A. This strain secretes an inactivated toxoid form of the C.perfringens alpha-toxin. Because this strain no longer expresses activealpha-toxin yet retains a significant portion of the toxin'santigenicity, it will be useful as a vaccine to protect against diseasecaused by C. perfringens.

EXAMPLE 3 Construction of Clostridium perfringens Recombinant CPERF002

In order to compensate for the lower transformation efficiency of C.perfringens strain 29 (see Table 1, supra) a new homology vector wasconstructed. The new vector incorporated C. perfringens sequences cloneddirectly from the strain 29 genome. This was predicted to result in amore efficient recombination step. The new vector was 1192-38.3 createdas follows.

In the first step the E. coli replication region and the C. perfringensresistance genes were cloned from shuttle plasmid pJIR418 (Sloan et al,Plasmid 27, 207 (1992); Genebank M77169). Plasmid pJIR418 was digestedwith restriction enzyme Ndel. Ligation of the large fragment producedplasmid 1192-23.1. This plasmid lacks a C. perfringens origin ofreplication, but unlike the plasmid 1162-45.1 that was constructed inExample 1, it retains the entire multiple cloning site of pJIR418

In the next step the C-terminal half of the plc gene was sub-cloned intothe intermediate plasmid 1192-23.1. Genomic DNA from strain 29 was usedas a template for long range PCR. The region of the plc gene(alpha-toxin) from the Bam HI site through a portion of the CPE0038 genewas amplified. The upstream plc primer, 5′CTGGGATCCTGATACAGATAATAATTTCTCAAAGGAT 3′ (SEQ ID NO: 18) corresponds tonucleotides 48880-48916 of strain 13 (Genbank NC 003366). The downstreamprimer within the CPE0038 gene, 5′actctgcagTTGTCATATCAATTAAATTAACTATAATCCC 3′ (SEQ ID NO: 19) iscomplementary to nucleotides 51244-51275 of strain 13 and containsflanking nucleotides including a Pst I restriction site (lowercase). Aproduct of 2402 base pairs was obtained and digested with restrictionenzymes Bam HI and Pst I. This fragment was ligated with the largefragment of Bam HI and Pst I digested 1192-23.1 to produce plasmid1192-36.10.

In the final step the N-terminal half of the plc gene was cloned viaPCR. The upstream primer, 5′ actgagctcCTAGACACTTTGCTTCAATATTTGGGAA 3′(SEQ ID NO: 20) corresponds to nucleotides 46513-46540 of strain 13 andincludes flanking nucleotides and a Sac I site (lowercase). Thedownstream primer, 5′ actggatccGCATTCTTATCATAATCTGGATAAGTAGAACC 3′ (SEQID NO: 21) is complementary to nucleotides 48824-48855 of strain 13 andincludes flanking nucleotides and a Bam HI site. A 2363 base pairproduct was produced and this was digested with Sac I and Bam HIrestriction enzymes. The digested fragment was ligated with the largefragment of Sac I and Bam HI digested 1192-36.10 to produce plasmid1192-38.3. Subsequent sequencing of the region flanking the plc Bam HIsite in 1192-38.3 confirmed the deletion of nine amino acids from tyr 62through trp 70.

Plasmid 1192-38.3 was used to electroporate C. perfringens strain 29cells. Strain 29 was previously shown to transform with an efficiency of3.6×104 transformants/μg of pJIR418 plasmid DNA. Strain 29 cells weregrown and electroporated as in Example 2 with the exception that aliquid selection technique was used after the three hour recoveryperiod. Instead of plating, 0.67 ml aliquots of cells were diluted into12 ml of TSYC+25 μg/ml chloramphenicol and grown overnight at 37° C. inan anaerobic jar. This modification was used because of the lowertransformation and plating efficiencies of strain 29 versus 1240. Afterovernight growth, cells were diluted again in selection media. Cellsfrom the second grow out were then passed five times without selectionprior to plating on blood agar plates. None of the colonies from theblood agar plates were non-hemolytic. Two blood plates were then replicaplated to TSYC, TSYC+25 μg/ml chloramphenicol and TSYC+50 μgerythromycin plates. All colonies were erythromycin sensitive but onlyone of 130 colonies was chloramphenicol sensitive. This colony,1192-45.4B, was renamed CPERF002. PCR analysis of genomic DNA fromCPERF002 with alpha-toxin specific primers showed a positive band foralpha-toxin. This band was smaller than the corresponding band fromwild-type strain 29 DNA. Primers specific for the chloramphenicol anderythromycin resistance genes failed to amplify CPERF002 DNA but showedstrong positive bands from plasmid 1192-38.3. Sequencing of the CPERFOO2alpha-toxin confirmed the nine amino acid deletion (see FIG. 3).

CPERF002 was assayed for expression of the inactivated alpha toxoidprotein. After 6 hours of anaerobic growth, aliquots of 1 ml of cellswere collected and centrifuged. Fifteen microliters of theunconcentrated supernatant media were analyzed by polyacrylamide gelelectrophoresis and subjected to Western Blot analysis with a rabbitpolyclonal antibody directed against recombinant alpha-toxin protein(Vaccine 11: 1253 (1993)). The results showed specific antibodyreactivity with a protein of the expected size for the alpha toxoidprotein.

CPERF002 is a genetically engineered deletant strain of C. perfringensType A. This strain secretes an inactivated toxoid form of the C.perfringens alpha-toxin. Because this strain no longer expresses activealpha-toxin yet retains a significant portion of the toxin'santigenicity, it will be useful as a vaccine to protect against diseasecaused by C. perfringens.

EXAMPLE 4 C. perfringens Deletant Vaccines

The CPERF001 and CPERF002 deletant vaccine strains described in Examples2 and 3 were evaluated for their ability to provide protection againstchallenge with wild-type C. perfringens. The first part of the study wasdesigned to determine if the administration of the live vaccine strainshad any adverse effect on the hatchability of embryonated eggs. Theassignment of experimental groups and the safety results are describedby Tables 2 TABLE 2 SAFETY RESULTS Number of Eggs Group* Vaccine(dose)** Hatched (%) 1 CPERF001 (0.8 × 10²)^(x) 18 (90%) 2 CPERF001 (0.8× 10³) 17 (85%) 3 CPERF001 (0.8 × 10⁴) 11 (55%) 4 CPERF001 (0.8 × 10⁵)16 (80%) 5 CPERF002 (1.9 × 10²) 16 (80%) 6 CPERF002 (1.9 × 10³) 17 (85%)7 CPERF002 (1.9 × 10⁴) 14 (70%) 8 CPERF002 (1.9 × 10⁵) 12 (60%) 9 Strain1240 (1.5 × 10³) 16 (80%) 10 Strain 29 (3.7 × 10³) 13 (65%) 11 MediaControls 19 (95%) 12 Uninoculated Controls 18 (90%)*20 eggs per group**100 μ1 dose given in ovo at 18 days of embryonation (IM)^(x)Titer per dose is measured as colony forming units (cfu″)

At a dose of 10³ or less (groups 1,2, 5, and 6), hatchability in thesegroups was not significantly lower than in the group inoculated withmedia only (group 11) or the group that was not inoculated (group 12).At the lowest dose CPERF001 showed the same hatchability as the mediacontrol and better than the uninoculated control group.

Because the higher doses of the deletants may have had an adverse effecton the hatchability of the eggs, only the two lowest dosage groups foreach deletant were included in the efficacy portion of the study. Thesegroups, along with the media control birds (group 11), were challengedwith (wild-type) C. perfringens strain CP6 that was administered orallyat approximately 10⁸ cfu/ml/bird when the birds were 20, 21 and 22 daysof age. All birds were necropsied at 25 days of age and lesions withinthe small intestine were scored using the rating scale for necroticenteritis summarized below. Necrotic Necrotic Necrotic Necrotic 0 ScoreEnteritis 1+ Enteritis 2+ Enteritis 3+ Enteritis 4+ No NE gross Thin andflaccid Single or few Extensive Dead animal lesions on small intestinalwall multifocal areas multifocal areas with NE gross intestine;(intestine of reddening of necrosis and lesions scored intestine hasremains flat and swelling of ulceration of the 2+ or above. normalelasticity when opened the intestinal intestinal (rolls back on anddoesn't roll wall; single or mucosa ± itself after being back into fewmultifocal significant opened). normal areas of hemorrhage or position);ulceration of layer of fibrin or excess or necrosis of the necroticdebris thickened intestinal on the mucosal mucus covering mucosa.surface (Turkish mucus towel membrane or appearance). focal ormultifocal mild reddening of the mucosa or congestion of the serosalvessels.Scoring with minor modifcations is according to Charles Hofacre, D.V.M.,M.A.M., Ph.D., University of Georgia, Poultry Diagnostic and ResearchCenter, 953 College Station Road, Athens, GA 30602,

Five (5) birds from the unvaccinated control group (not challenged) werenecropsied at the end of the study to confirm there was no exposure toC. perfringens during the course of the study. The results are presentedin Table 3. TABLE 3 TREATMENT GROUPS FOR PROTECTION* RESULTS In-ovoVaccine Days of age at Challenge Group* Vaccine Dose (cfu) with C.perfringens N Mean 1 CPERF001 1 × 10² 20, 21 and 22 9 0.67 2 CPERF001 1× 10³ 20, 21 and 22 13 0.46 5 CPERF002 1 × 10² 20, 21 and 22 12 1.33 6CPERF002 1 × 10³ 20, 21 and 22 12 1.08 11 Media None 20, 21 and 22 151.80 control 12 None None Not challenged 5 0.00*necropsy at 25 days of age

Scores for Groups 1 and 2 were each statistically significant lowercompared to Groups 11 (Wilcoxon Exact Rank Sum Test p≦0.0250). Meanscores for Groups 5 and 6 were lower than Group 11 but not statisticallydifferent (Wilcoxon Exact Rank Sum Test p≧0.2177). An estimate ofvaccine efficacy was performed according to the procedure described byDavid Siev [Journal of Modern Applied Statistical Methods Vol. 4, No. 2,500-508 (2005)]. Vaccine efficacy in reducing disease severity wasestimated at 54% for Group 1, 65% for Group 2, 20% for Group 5 and 27%for Group 6 when compared to Group 11.

EXAMPLE 5 Construction of a C. perfringens Swine Deletant

The strategies used in Examples 1 and 2, supra, are used to constructalpha-toxin deletion mutants from a swine strains of C. perfringens.Initially, field isolates from diseased swine are electroporated withplasmid pJIR418 to assess their transformability. Isolates yielding ≧10⁴transformants per microgram of plasmid DNA and susceptible to eitherchloramphenicol or erythromycin are candidates for deletion.

Genomic DNA from the candidate strains is used as a template for longrange PCR of the plc (alpha-toxin) gene and flanking sequences. Aftersubcloning of the PCR products, the alpha-toxin gene and flankingregions are sequenced and restriction mapped. New oligonucleotideprimers are synthesized with flanking restriction sites and the productsof two separate amplifications are cloned into the suicide plasmid1192-23.1 to create the 27 base pair deletion as in Example 1.

The swine suicide vector is electroporated into the corresponding swinestrain of C. perfringens and deletion mutants are isolated using themethods described in Example 2, supra. Deletion mutants are confirmed bythe absence of beta hemolysis on blood agar plates and by DNA sequencingof the alpha-toxin gene.

This construct is a genetically engineered deletant strain of C.perfringens Type A. This strain secretes an inactivated toxoid form ofthe C. perfringens alpha-toxin. Because this strain no longer expressesactive alpha-toxin yet retains a significant portion of the toxin'santigenicity, it is useful as a vaccine to protect swine against diseasecaused by C. perfringens.

Biological Deposit

Cultures of the following biological materials have been deposited withthe following international depository:

American Type Culture Collection (ATCC) 10801 University Boulevard,Manassas, Va. 20110-2209, U.S.A., under conditions that satisfy therequirements of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure.Organism Accession No. Date of Deposit Clostridium perfringens PTA7364Feb. 7, 2006 CPERF/ΔαToxin 365-054 Clostridium perfringens PTA7365 Feb.7, 2006 CPERF/ΔαToxin 365-053

1. A nucleic acid molecule that encodes a substantially nontoxic muteinof Clostridium perfringens alpha-toxin; wherein the mutein alpha-toxincomprises the amino acid sequence of SEQ ID NO: 3 minus at least 9consecutive amino acid residues; and wherein one of the deleted aminoacid residues is His₆₈.
 2. The nucleic acid molecule of claim 1 whereinthe mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3minus at least 12 consecutive amino acid residues; and wherein one ofthe deleted amino acid residues is His₆₈.
 3. The nucleic acid moleculeof claim 2 wherein the mutein alpha-toxin comprises the amino acidsequence of SEQ ID NO: 3 minus at least 18 consecutive amino acidresidues; and wherein one of the deleted amino acid residues is His₆₈.4. The nucleic acid molecule of claim 1 wherein the mutein alpha-toxincomprises SEQ ID NO: 3 minus 9 consecutive amino acid residues rangingfrom Tyr₆₂ through Trp₇₀.
 5. The nucleic acid molecule of claim 1comprising the nucleic acid sequence of SEQ ID NO: 2; whereinnucleotides 268-294 of the nucleic acid molecule are deleted.
 6. Thenucleic acid molecule of claim 5 wherein the deleted nucleotides arereplaced by a nucleotide sequence encoding a single Leu residue.
 7. Asubstantially nontoxic mutein of Clostridium perfringens alpha-toxin;wherein the mutein alpha-toxin comprises the amino acid sequence of SEQID NO: 3 minus at least 9 consecutive amino acid residues; and whereinone of the deleted amino acid residues is His₆₈.
 8. The substantiallynontoxic mutein of Clostridium perfringens alpha-toxin of claim 7wherein the mutein alpha-toxin comprises the amino acid sequence of SEQID NO: 3 minus at least 12 consecutive amino acid residues; and whereinone of the deleted amino acid residues is His₆₈.
 9. The substantiallynontoxic mutein of Clostridium perfringens alpha-toxin of claim 8,wherein said mutein comprises the amino acid sequence of SEQ ID NO: 3minus at least 18 consecutive amino acid residues; and wherein one ofthe deleted amino acid residues is His₆₈.
 10. The substantially nontoxicmutein of Clostridium perfringens alpha-toxin of claim 7, wherein nineconsecutive amino acid residues that range from Tyr₆₂ through Trp₇₀, aredeleted and replaced by a single Leu residue.
 11. An attenuatedClostridium perfringens organism constructed by integrating the nucleicacid molecule of claim 1 into a chromosome of a non-attenuatedClostridium perfringens organism.
 12. The attenuated Clostridiumperfringens organism of claim 11 that is substantially nontoxic.
 13. Theattenuated Clostridium perfringens organism of claim 11 wherein thenucleic acid molecule is located at a position on the chromosome that ishomologous to the location of a nucleic acid molecule encoding awild-type alpha-toxin present in the non-attenuated Clostridiumperfringens.
 14. The attenuated Clostridium perfringens organism ofclaim 11 that is a type A Clostridium perfringens.
 15. The attenuatedClostridium perfringens organism of claim 11 wherein the non-attenuatedClostridium perfringens from which it was constructed was isolated froma host animal that is either a mammal or an avian.
 16. The attenuatedClostridium perfringens organism of claim 15, wherein said mammal isselected from the group consisting of bovine, ovine, and porcine. 17.The attenuated Clostridium perfringens organism of claim 15 wherein saidavian is a chicken, a turkey, a goose, a duck, a swan, a dove, a pigeon,a grouse, or a partridge.
 18. A vaccine comprising the attenuatedClostridium perfringens organism of claim
 11. 19. The vaccine of claim18 further comprising one or more of the following: a pharmacologicallyacceptable buffer, excipient, or an adjuvant.
 20. A method of inducingimmunity to Clostridium perfringens in an animal, comprisingadministering to the animal an immunologically effective dose of thevaccine of claim
 18. 21. The method of claim 20 wherein the vaccine isadministered by one or more of the following routes: oral,intramuscular, intravenous, intradermal, subcutaneous, or intranasal.22. The method of claim 20 wherein the vaccine is top-dressed on thefeed of the animal.
 23. The method of claim 20 wherein the vaccine issprayed onto the animals to provide for oral administration.
 24. Ananimal feed that comprises the vaccine of claim
 18. 25. The attenuatedClostridium perfringens organism of claim 11 that comprises at least onegene encoding a non-Clostridium perfringens polypeptide.
 26. Theattenuated Clostridium perfringens organism of claim 25 wherein saidnon-Clostridium perfringens polypeptide is a non-bacterial polypeptide.27. The attenuated Clostridium perfringens organism of claim 26 whereinthe non-bacterial polypeptide is an avian or mammalian polypeptide. 28.The attenuated Clostridium perfringens organism of claim 27 wherein thenon-bacterial polypeptide is an avian polypeptide.
 29. The attenuatedClostridium perfringens organism of claim 27 wherein the non-bacterialpolypeptide is a cytokine.
 30. The attenuated Clostridium perfringensorganism of claim 29 wherein the cytokine is chicken IL-18.
 31. Anantibody that selectively binds to an epitope found in Clostridiumperfringens alpha-toxin but missing from the substantially nontoxicmutein of Clostridium perfringens alpha-toxin of claim 11 wherein saidantibody can distinguish said substantially nontoxic mutein from a wildtype Clostridium perfringens alpha-toxin.
 32. A test kit for identifyingwhether a subject animal has been naturally infected by a Clostridiumperfringens organism comprising the antibody of claim
 31. 33. A methodof identifying an animal that has been naturally infected by aClostridium perfringens organism from one vaccinated with an attenuatedClostridium perfringens organism, said method comprising: (a) contactinga fluid sample from the animal with the antibody of claim 31, (b)determining whether the antibody reacts with the fluid sample; whereinwhen the antibody reacts with the fluid sample, the animal is identifiedas one that has been naturally infected by a Clostridium perfringensorganism.
 34. A Clostridium perfringens organism that is CPERF/ΔαToxin365-054 having ATCC Deposit No. PTA7364.
 35. A Clostridium perfringensorganism that is CPERF/ΔαToxin 365-053 having ATCC Deposit No. PTA7365.